Cassette and embedding assembly for handling and holding tissue samples during processing, embedding and microtome procedures, staging devices therefore, and methods therefor

ABSTRACT

The disclosure relates to a cassette, frame and mold for holding a tissue sample during an embedding and mircotoming process, and related methods. The cassette is sectionable in a microtome and includes a body with a bottom wall and a plurality of side walls. First and second side walls are generally V-shaped to present an apex of the “V” to the microtome blade. A lid of the cassette is stiffer than the bottom wall of the cassette to assist with positioning the tissue sample. The side walls of the cassette are perforated so as to significantly reduce the amount of cassette material that must be cut by the microtome blade. In one embodiment, to additionally reduce blade wear the ribs on one side wall are offset lengthwise relative to the ribs on an opposite side wall. An upper flange of the cassette includes depressions configured to register with detents in the frame.

The present application is a continuation of PCT Serial No.PCT/US02/30775 filed on Sep. 26, 2002 (now pending), the disclosure ofwhich is hereby fully incorporated by reference herein.

FIELD OF THE INVENTION

The present invention generally relates to supports for handling andembedding tissue samples for pathological analysis and, moreparticularly, to cassettes which can receive one or more tissue samplesand be embedded and subsequently microtomed with the tissue sample orsamples.

BACKGROUND OF THE INVENTION

To accurately diagnose various tissue diseases and conditions, medicalpersonnel must remove one or more samples of tissue from the body of apatient. This process of harvesting tissue from the body is known as abiopsy. Once the tissue sample or samples are removed and sent to apathology laboratory, the tissue will go through a series of proceduresperformed by a histotechnician and, ultimately, a pathologist, in orderto diagnose the tissue. The present invention generally relates to thoseprocedures that are normally performed by the histotechnician to preparethe tissue sample or samples into slides that may be analyzed under amicroscope by the pathologist.

Although the singular term “sample” is used throughout thisspecification, it should be understood that this term likewiseencompasses plural “samples” as well. Once a tissue sample is removedfrom the body of a patient, it is typically placed into a specimencontainer containing a tissue fixative solution and then the containeris transported to a pathology laboratory. The tissue will undergo aprocess known as “grossing-in” in the pathology lab during which ahistotechnician will retrieve the tissue sample from the container,typically cut the tissue into appropriate sizes for tissue processing,place individual samples into the appropriate sized small plastic tissuecassettes, and assign tracking numbers to each cassette. These trackingnumbers are then logged into a tracking system used in the laboratory.For the smallest tissue samples, which may only be scrapings, thecassette will have fine mesh openings on the sides and bottoms. In othersituations involving very small tissue samples, the samples are placedinto a bag that resembles a tea bag and prevents the smallest tissuesamples from escaping. Larger tissue samples are placed into cassetteshaving somewhat larger slotted openings which are again smaller than thetissue sample inside the cassette.

The cassettes are then placed into a stainless steel perforated basketand run through a tissue processing machine, often overnight. Thismachine uses a combination of vacuum, heat, and chemicals to remove theinterstitial fluids. Once the fluids have been removed from the tissuesamples, the processing machine immerses the tissues samples in a bathof molten paraffin so that the interstices in the tissue are replacedwith paraffin. The histotechnician then removes the basket from themachine and removes the individual tissue cassettes. At an embeddingstation, which has a molten paraffin reservoir and dispenser, thehistotechnician will individually remove the tissue from each cassette.The histotechnician must carefully orient the tissue sample, based ontissue type, into a stainless steel base mold which is roughly the sizeof the tissue cassette and is partially filled with molten paraffin. Thetissue sample must be manually held, typically using forceps, againstthe bottom of the mold. If it is not, this could compromise the abilityto make proper slices of the tissue later in the microtome. The moltenparaffin is then rapidly cooled on a refrigerated plate, which may be athermal electric cooler (TEC), to partially solidify the paraffinthereby holding the tissue sample in the proper orientation against thebottom of the mold. The cassette is then placed on top of the base moldand paraffin is poured through the opened top of the cassette into thebase mold. The cassette changes its function at this point in theprocedure from a tissue holding component to a fixation device for lateruse in taking shavings or slices from the solidified paraffin in amicrotome. The base mold is chilled until all of the molten paraffin hassolidified and the histotechnician removes the stainless steel base moldfrom the block of embedded paraffin. The tissue sample is thus embeddedwithin a rectangular block of paraffin with a plastic tissue cassette onthe opposite side which will then be used as a holder in the chuck ofthe microtome. As with the tissue processing machine, the embeddingprocess is accomplished in a batch fashion during which an averagehistotechnician may embed approximately 40 to 60 cassettes per hour.

The blocks of hardened paraffin containing the embedded tissue samplesare then ready to be sliced into extremely thin sections for placementon a microscope slide. The histotechnician mounts the embedded tissueblock in a chuck on the microtome which is sized to accept the side ofthe block that has the embedded plastic cassette. The histotechniciancan then begin slicing the paraffin block which has the tissue sampleembedded opposite to the plastic cassette surface. This yields a ribbonof individual slices of the tissue embedded in the paraffin. The actionof the microtome causes the individual slices to stick together whendone properly and, subsequently, these very thin ribbons of slices arefloated into a water bath and a glass slide is carefully placedunderneath the slice. The slice, with the thin sectioned tissue sampleembedded therein, is then adhered to the top of the slide.

When the histotechnician has enough slides from the tissue sample, theslides are placed into an automatic staining machine. The stainingmachine goes through a series of infiltrating steps to stain thedifferent tissue and cells of the slide different colors. This helps thepathologist identify different structures and makes it easier to findany abnormalities in the tissue. After the staining procedure iscomplete, the slides are cover slipped and prepared for the pathologistto place under a microscope to analyze.

Based on the summary of the procedure provided above, it will beappreciated that conventional tissue sample handling and processing is avery labor-intensive process involving several manual steps performed bya histotechnician. Thus, repetitive stress injuries such as carpaltunnel syndrome are prevalent. This is especially true with the tissuesample embedding process. These multiple manual operations and repeatedtissue handling increase the likelihood of human error and, moreover,require highly trained and skilled histotechnicians to ensure that thetissue samples ultimately adhered to the slides for analysis by thepathologist are in an optimum condition and orientation to make accuratediagnoses.

U.S. Pat. No. 5,817,032 (the '032 patent) discloses various improvementsto this area of technology, including new manners of holding tissuesamples during the grossing in, embedding, and microtome or slicingprocedures. More specifically, the '032 patent relates to a tissuetrapping and supporting device, which may be a cassette, and which maybe cut with a microtome. When a cassette is used, the tissue sample isimmobilized within the cassette and subjected to the process forreplacing tissue fluids with paraffin. Then, the tissue sample and thecassette are sliced at the same time for mounting on microscope slides.Because the tissue sample is never removed from the cassette from thetime it is processed in the tissue processing machine to the time thatit is cut with the microtome, a significant amount of handling time issaved. Moreover, the chance for human error or tissue loss due, forexample, to dropping the tissue during handling, is significantlyreduced due to the elimination of separate tissue handling steps. Thispatent also generally discusses an automated process which, inconjunction with the novel tissue cassettes, even further reduces thehandling steps during the entire procedure.

In spite of the various advances made in this field, there is anincreasing need for additional improvements related to increasedproduction capability and increased and more consistent quality ofembedded tissue samples and resulting slices or ribbons of embeddedtissue which will be subject to diagnosis.

SUMMARY OF THE INVENTION

Generally the invention relates to a cassette for holding a tissuesample during an embedding and microtoming or slicing process. Thecassette includes a body with a bottom wall and a plurality of sidewalls extending upwardly with respect to the bottom wall to define aninterior space for receiving the tissue sample. The bottom wall and theplurality of side walls are constructed of a material capable of beingsectioned in a microtome. Preferably, the cassette material is alsoresistant to any type of degradation during processing which wouldcompromise its ability to function in accordance with the invention. Ina first aspect of the invention, the plurality of side walls comprisefirst and second side walls on opposite sides of the bottom wall eachincluding portions angling from approximate midpoints of the first andsecond side walls toward the other of the first and second side walls onthe opposite side of the bottom wall. In the preferred embodiment, thetwo longest side walls of the four side walls comprising a rectangularcassette are generally V-shaped in a direction away from the interior ofthe cassette. This presents an apex of the V-shape to the microtomeblade, after the embedding process is complete, which assists with thecutting action. Specifically, this feature has been found to reduce orprevent the hardened paraffin from breaking or cracking away from thecassette side wall material while making slices in the microtome.

The cassette preferably further includes a lid configured to be coupledwith the body for movement between open and closed positions. The lidmay be depressed downwardly on top of the tissue sample in the cassetteinterior. The lid is preferably stiffer than the bottom wall of thecassette. This feature allows the lid to position the tissue sample inthe cassette parallel to the bottom of the mold during the embeddingprocess. More specifically, the stiffer lid pushes the tissue sample andthe more flexible bottom wall of the cassette against the rigid bottomof a base mold while the molten paraffin solidifies. This helps ensurethat the bottom wall of the cassette can be removed in its entiretyduring a facing operation in the microtome prior to slicing the tissuesample, and that the tissue is positioned flatly against the bottom wallof the cassette.

In another aspect of the invention, the side walls of the cassette areperforated such that there is at least a ratio of about 2.5:1 of openarea to solid material area whereby the solidified paraffin occupies theopen area of the side walls. This ratio may be altered by usingdifferent materials for the cassette and/or paraffin embedding media.For instance, a higher molecular weight paraffin or lower moneluculerweight cassette will allow the ratio to vary somewhat. Currently, anindustry standard paraffin (e.g., Sakura VIP processing/embeddingmedium) works best with a ratio of at least about 3.0:1 and, morepreferably, at least about 3.5:1. This reduces the amount of cassettematerial that must be cut by the microtome blade while taking slices ofthe tissue and, therefore, increases blade life and quality of theresulting ribboned, embedded tissue samples. In addition this ratioassures that the paraffin is strong enough not to fracture when cut bythe microtome blade. Another feature that results in similar advantagesinvolves forming the side walls from ribs and offsetting the ribs of oneof the first and second side walls with respect to those of the oppositeside wall along the side wall length. Thus, the microtome blade willcontact a more uniform amount of the cassette material along its lengthduring each pass. This significantly reduces blade wear while cuttingthrough the cassette material. Reduced blade wear is advantageous tokeeping blade costs under control as the majority of blades used aredisposable.

In another aspect of the invention, the cassette further includes aflange extending along upper portions of at least two of the side walls.The flange includes depressions configured to register with detents in aframe during the tissue embedding process. This increases the effectiveheight dimension of the cassette interior, thereby allowing more tissueto be placed in the cassette and more passes to be made in themicrotome. In this regard each pass of the microtome may only take a 5micron slice. Therefore, using depressions having a depth, for example,of 0.14″ can allow about 70 more slices to be taken in the microtome.

The invention further contemplates the various unique assemblies of twoor more of the tissue cassette, frame, and base mold as disclosedherein. With respect to the frame and base mold, for example, structureis provided to physically hold the frame against the base mold. In thepreferred embodiment, a seal is provided to perform this holdingfunction and also to prevent leakage of liquid paraffin from the basemold.

In another aspect, the invention contemplates a device for staging atissue sample cassette from an upper position in a frame used during atissue embedding process to a lower position in the frame. The deviceincludes a handle, a staging mechanism coupled with the handle andconfigured to engage an upper surface of the cassette, and a stopoperatively coupled to the handle and configure to stop verticalmovement of the staging mechanism when the cassette reaches the lowerposition in the frame. The staging mechanism further comprises aplurality of fingers which are configured to engage a correspondingplurality of locations on the upper surface of the cassette. Forexample, four fingers may be provided for engaging four corner locationson the cassette. This helps ensure that the cassette is engaged with atleast four pairs of detents located proximate corner portions of thecassette thereby positioning the bottom wall of the cassette parallel toand against the bottom wall of the base mold.

In one embodiment, the staging device is a rigid member and the stopcomprises a fixed stop member coupled for movement with the stagingmechanism and configured to stop against an upper surface of the frame.In another embodiment, the device includes a stabilizing mechanismcoupled with the handle and moveable relative to the staging mechanism.The stabilizing mechanism is configured to engage an upper surface ofthe frame as the staging mechanism moves the cassette from the upperposition to the lower position within the frame. In this embodiment, thestaging mechanism is normally spring-biased into an upward position andis forced downward against the spring bias when moving the cassette fromthe upper position to the lower position. The stop in this embodimentfurther comprises respective surfaces of the handle and stabilizingmechanism which engage one another when the staging mechanism has placedthe cassette into the lower position. The staging devices of thisinvention ensure that the cassette is fully staged into the base mold,while ensuring that the cassette is not pushed too far through theframe. Moreover, the staging devices ensure that the bottom wall of thecassette and, therefore, the tissue sample, lay flat against the bottomof the base mold. This improves the efficiency and quality of tissueshavings later made in the microtome.

The invention also encompasses the various methods of using the tissuecassette and cassette/frame/base mold assemblies as disclosed herein.

These and other objects, advantages, and features of the invention willbecome more readily apparent to those of ordinary skill in the art uponreview of the following detailed description taken in conjunction withthe accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exploded perspective view of an assembly including thetissue cassette, the frame into which the cassette is inserted and thebase mold into which the frame and cassette assembly are inserted.

FIG. 2 is an assembled perspective view of the tissue cassette, frameand base mold.

FIG. 3 is a cross sectional view taken along line 3-3 of FIG. 2 andshowing the tissue cassette in its initial upper position.

FIG. 4 is an assembled perspective view, similar to FIG. 2 butillustrating the second, staged position of the tissue cassette.

FIG. 5 is a cross sectional view taken along line 5-5 of FIG. 4 andshowing the tissue cassette staged down into its second, lower positionagainst the bottom of the base mold.

FIG. 6 is a cross sectional view of the frame and the tissue cassetteembedded in material such as paraffin, after release from the base mold.

FIG. 7 is a perspective view of a manual, spring-biased staging device.

FIG. 8 is a longitudinal cross sectional view of the staging device ofFIG. 7 being used to stage a tissue cassette through the frame and intothe base mold.

FIG. 9 is a perspective view of a tissue cassette constructed inaccordance with a second embodiment of the invention.

FIG. 10 is a cross sectional view taken along line 10-10 of FIG. 9.

FIG. 11 is a perspective view of a manual staging device constructed inaccordance with a second embodiment.

FIG. 12 is a bottom perspective view of the staging mechanism associatedwith the staging device of FIG. 11.

DETAILED DESCRIPTION

Turning first to FIGS. 1-3, a tissue cassette 10 constructed inaccordance with the invention is received within a frame 12 and thetissue cassette 10 and frame 12 are then positioned in a base mold 14.After a staging operation the base mold 14 is subsequently filled withliquid paraffin, as described further below. Tissue cassette 10 includesa perforated body 20 formed by four side walls 22 a, 22 b, 22 c, 22 dand a bottom wall 24. Each wall is preferably constructed withperforations or openings 26 and ribs 28. An upper flange 30 surroundsand extends outwardly from the respective side walls 22 a-d. A lid 32 isattached to body 20 by a hinge 34 which allows vertical movement of lid32 into the interior of body 20 for retaining one or more tissue samplesagainst bottom wall 24. Lid 32 is likewise formed with perforations 36which may be elongated and generally separated by ribs 38. As shown bestin FIG. 1, elongated perforations 26, 36 on the bottom wall 24 and lid32 respectively extend toward central areas 24 a, 32 a of the bottomwall 24 and lid 32. This assists with allowing a mold (not shown) tofill with material, such as PFA, during the molding process of cassette10.

Lid 32 is formed with a shape along its circumference which complimentsthe shape of side walls 22 a-d. In this regard, respective lengthwiseside edges 40 a, 40 b of lid 32 are formed in the shape of a shallow “V”and each includes an apex 42 a, 42 b generally at the central portionthereof. This likewise corresponds to the lengthwise shallow “V” shapeof side wall 22 a and opposite side wall 22 c. Thus, when the embeddedtissue cassette is later placed in a microtome and sections are slicedfrom the embedded tissue cassette, the microtome blade will initiallycontact the apex corresponding of side wall 22 a or 22 c, depending onwhich side is facing up in the microtome. This has been found toincrease the quality of ribboned slices being made from the embeddedtissue cassette. That is, little or no fracturing of paraffin will occurat the paraffin/cassette interfaces.

Frame 12 more specifically includes an open interior 50 which receivescassette 10, and an angled front wall 52 which may be used for recordingindicia, such as patient data. Respective upper and lower sets ofdetents 54, 56 and 58, 60 extend inwardly into the open interior 50 offrame 12. Initially, tissue cassette 10 is held between the upper pairsof detents 54, 56 by pressing the upper flange 30 downwardly past theupper sets of detents 54 and against the lower set of detents 56 afterone or more tissue samples have been placed into cassette body 20 andlid 32 has been closed. Respective detents 61 extend inwardly from sidewalls 22 a-d to allow lid 32 to be snapped and retained in a closedposition. During a staging operation, tissue cassette 10 will be movedvertically downwardly through frame 12 until flange 30 snaps pastdetents 58 and rests against detents 60 at a lower position (FIG. 5).Recesses 62, 64 are formed in at least two opposite side walls forallowing fingers of a gripper mechanism to register with the assemblyduring automated handling and embedding operations, as necessary.Preferably side walls 66 a, 66 b contain these recesses 62, 64, whileadditional side walls 66 c, 66 d may include additional structureallowing automated handling, or allowing other functions as necessary.Base mold 14 includes an open interior 70 for receiving frame 12 and maybe surrounded by a resilient elastomeric seal 72 which prevents theescape of liquid paraffin during the embedding process. This eliminatesan additional step of scraping off excess hardened paraffin from frame12 after the embedding process is complete. Such excess paraffin canprevent the frame from properly fitting in a microtome chuck.

By comparing FIGS. 2 and 3 to FIGS. 4 and 5, it will be appreciated thatduring the staging process, tissue cassette 10 is moved verticallydownward into the interior 70 of base mold 14 such that the bottom 24 oftissue cassette 10 contacts the bottom 74 of base mold 14. At thisposition, flange 30 of tissue cassette body 20 is received betweenrespective detents 58, 60 holding it in this lower position. Frame 12 ispreferably press fit and held frictionally within base mold 14 by one ormore resilient seals 72. Seal 72 performs two functions in thisembodiment. First, it physically holds frame 12 within base mold 14 byfriction. This prevents the frame 12 and attached cassette 10 fromfloating or otherwise moving while base mold 14 is filled with paraffin.Second, it prevents the liquid paraffin from escaping from base mold 14in the area between outer walls 12 a of frame 12 and interior 70 of basemold 14. It will be appreciated that a holding member other than seal 72may be used instead to physically hold frame 12 against base mold 14. Inthis case, seal 72 may not be necessary. As some examples, such holdingmembers may be clamps, fasteners, spring members or weights. In thesefigures, the tissue sample or samples within cassette 10 have beendeleted for clarity. However, it should be understood, that lid 32 willbe depressed down onto one or more tissue samples 80 contained with theinterior of cassette body 20 (FIG. 6). While the assembly of cassette10, frame 12 and base mold 14 is in the staged configuration shown inFIGS. 4 and 5, liquid paraffin is introduced through the open interiorof frame 12 and through the perforations 26, 36 of cassette 10 into theinterior 70 of base mold 14. The liquid paraffin is then preferablycooled on a suitable cooling device, such as a TEC, and the assembly ofthe tissue cassette 10, frame 12, embedded tissue sample 80 and hardenedparaffin 82 is removed from base mold 14 as shown in FIG. 6. Frame 12may then be used as a device to fix the assembly in a microtome chuckand slices are then taken from the bottom surface 82 a of paraffin 82.Initially, a facing blade is used to remove the initial layer ofparaffin 82 and bottom wall 24 of tissue cassette 10. At this point, adifferent microtome blade may be used to take ribboned slices orshavings of tissue sample 80 and the surrounding paraffin 82 and sidewalls 22 a-d of tissue cassette body 20. Often times the same blade thatis used for facing may also be used for sectioning.

FIGS. 7 and 8 illustrate one type of staging device 100 which may beused in conjunction with the tissue cassette 10, frame 12 and base mold14 described above. Staging device 100 includes a handle 102 at an upperend and a staging mechanism 104 at a lower end. Staging mechanism 104 iscoupled to a hollow cylinder 106 extending upwardly into a shroud 108rigidly connected with handle 102. A plurality of four stabilizermembers 110 are rigidly coupled with cylinder 106, while a plurality ofpreferably movable staging fingers 112 are coupled to a reciprocatingshaft 114 which extends through cylinder 106. Shaft 114 is rigidlyconnected to handle 102 and is biased therewith in an upward position bya spring 116 positioned between a bottom surface 102 a of handle 102 andan upper surface 106 a of cylinder 106. Thus, it will be appreciatedthat stabilizer members 110 may be placed against an upper side of frame12 for stabilizing purposes and handle 102 may then be depresseddownwardly as indicated by arrow 118. This moves reciprocating shaft 114and the attached staging fingers 112 downwardly against tissue cassette10 thereby moving the tissue cassette 10 relative to frame 12 from itsupper position shown in FIG. 3 to its lower, staged position shown inFIG. 5. To avoid pushing cassette 100 too far through frame 12, surface108 a stops against surface 110 a as flange 30 reaches its lowerposition between detents 58, 60.

FIGS. 9 and 10 illustrate a second embodiment of a tissue cassette 120.Tissue cassette 120 includes a cassette body 122 having four side walls124 a-d surrounding an open interior and bounded on a bottom side by abottom wall 126. Side walls 124 a-d are constructed of ribs 128separated by perforations 130 and bottom wall 126 is constructed by ribs132 separated by perforations 134. Ribs 128 of side wall 124 a areoffset in a lengthwise direction relative to ribs 128 of opposite sidewall 124 b, as shown by distance “d” in FIG. 10. Distance “d” may vary,however, in this embodiment, on average, it is approximately 0.015″ to0.030″. Offsetting ribs 128 in this manner ensures that a microtomeblade passing through walls 124 a and 124 b contacts a more uniformamount of cassette material along its length. This leads to longer bladelife, more uniform blade wear and more consistent high quality slices ofembedded tissue. Ribs 132 and perforations 134 extend in theirlengthwise direction toward a central area 126 a of bottom wall 126. Inthis preferred embodiment, side walls 124 a-d are constructed such thatthe ratio of plastic cassette material, such as PFA, to the open areaformed by perforations 130 is approximately 3.7:1. To achieve at leastthis ratio in the preferred embodiment, ribs 128 have a width W1 ofapproximately 0.010″ to 0.014″ while perforations 130 have a width W2 ofapproximately 0.040″ to 0.050″. This perforated area, which is belowsolid side wall portion 136 is the area which will be cut with amicrotome blade after embedding of tissue sample 80 (FIG. 6). Especiallywhen using PFA with a Shore D hardness of 48 to 55 as the material forcassette 120, in combination with the industry standard paraffinembedding material mentioned above, this ratio of cassette material toopen area has been found to improve the quality of cuts made through theembedded cassette and increase blade life significantly. Depending onthe respective molecular weights of the embedding material and thecassette material, this ratio may be increased or reduced.

A flange 140 surrounds the upper side of cassette body 122 and containsrespective upwardly facing depressions 142 a, 142 b, 142 c, 142 d. Anumber of downwardly facing depressions 142 e, 142 f (only two ofseveral referenced) are also contained in flange 140. These depressionsrespectively register with at least detents 54, 56 and 58, 60 of frame12 (see FIGS. 3 and 5). It will be appreciated that additional lowerdetents may be formed on frame 12 to provide further support and preventcassette 10 from being pushed too far through frame 12. In such a case,additional depressions are formed in the underside of flange 140 toaccommodate the additional supportive detents. The depressions allow theeffective outward extension of cassette body 120 from frame 12 to beincreased thereby increasing the number of slices that may be taken fromthe embedded cassette in a microtome. This can be important in manysituations in order to obtain the desired slides for pathologicexamination.

Detents 144 are also formed on side walls 124 a-d for retaining a lid150 in place. Lid 150 is coupled to cassette body 122 by a hinge 152.Lid 150 is formed to be stiffer than bottom wall 126 of cassette body122 so that lid 150 may be used to press the tissue sample against thebottom wall 126 and to press the bottom wall 126 against the rigidbottom 74 of base mold 14 (FIG. 5). Lid 150 is formed by a plurality ofribs 154 generally separated by perforations 156 and each extending in alengthwise elongated fashion toward a central area 150 a of lid 150. Lid150 further includes side edges 158 a, 158 b extending lengthwisethereon and having a shallow “V” shape with a central apex 160 a, 160 b.Angle α (FIG. 10) is preferably about 4°. As with the first embodiment,the shape of side edges 158 a, 158 b complements similar shape of sidewalls 124 a, 124 b of cassette body 122. In this regard, these sidewalls 124 a, 124 b, either one of which may be the initial wall cut inthe microtome, include respective apecies 162 a, 162 b. Respective ends164 a, 164 b of lid 150 also complement the similar shape of cassettebody side walls 124 c, 124 d to achieve a close fit of lid 150 withincassette body 122. The use of cassette 120 in the process of embeddingone or more tissue samples, including the staging process within frame12 and the embedding or molding process within base mold 14 is the sameas described above with regard to FIGS. 1-8.

FIGS. 11 and 12 illustrate a manual staging device 200 in accordancewith a second embodiment. Staging device 200 comprises an upper handle202 and a lower staging mechanism 204 coupled together rigidly by ashaft 206. In FIG. 12, handle 202 and shaft 206 have been deleted forclarity. Staging mechanism 204 preferably comprises a plurality of fourstaging members 210, 212, 214, 216 extending generally radially outwardfrom shaft 206 and each including a finger portion 210 a, 212 a, 214 a,216 a. The finger portions 210 a-216 a respectively engage upper cornerportions of cassette 10 (FIG. 1) or cassette 120 (FIG. 9). When the userthen pushes downward on handle 202, finger portions 210 a-216 a urgecassette 10 or cassette 120 downward into the lower, staged position(FIG. 5). To prevent the cassette from being moved too far downward, apair of stops 218, 220 are provided on staging mechanism 204. In thisembodiment, stop surfaces 218 a, 220 a stop against the upper surface offrame 12 as cassette 10 or cassette 120 reaches the lower, stagedposition (FIG. 5). Thus, the distance between the lower ends of fingerportions 210 a-216 a and stop surfaces 218 a, 220 a equates to thedistance between upper detent pair 54, 56 and lower detent pair 58, 60.It will be appreciated that other forms of staging mechanisms and stopsmay be provided while falling within the spirit and scope of theinvention.

While the present invention has been illustrated by the description ofthe various embodiments thereof, and while the embodiments have beendescribed in considerable detail, it is not intended to restrict or inany way limit the scope of the appended claims to such detail.Additional advantages and modifications will readily appear to thoseskilled in the art. The invention in its broader aspects is thereforenot limited to the specific details, representative apparatus andmethods and illustrative examples shown and described. Accordingly,departures may be made from such details without departing from thescope or spirit of Applicant's general inventive concept.

1. A cassette for holding a tissue sample, comprising: a body includinga perforated bottom wall and a plurality of perforated side wallsextending upwardly with respect to said bottom wall to define aninterior space for receiving the tissue sample; said bottom wall andsaid plurality of side walls being constructed of a material capable ofbeing successfully sectioned in a microtome and resistant to degradationduring histologic tissue processing, wherein said plurality of sidewalls comprise first and second side walls each having a length andextending upwardly from opposite edges of said bottom wall, at least oneof said first and second side walls including portions angling towardthe other of the first and second side walls on the opposite edge of thebottom wall to define an outwardly projecting portion proximate amidpoint along said length of said at least one side wall.
 2. Thecassette of claim 1, further comprising a lid configured to be coupledwith said body and movable between open and closed positions, said lidbeing stiffer than said bottom wall.
 3. The cassette of claim 1, whereinsaid plurality of side walls comprise perforated walls in which theratio of open area to solid area is at least about 3.5:1.
 4. Thecassette of claim 1, wherein said plurality of side walls compriseperforated walls in which the ratio of open area to solid area is atleast about 3.0:1.
 5. The cassette of claim 1, wherein said plurality ofside walls comprise perforated walls in which the ratio of open area tosolid area is at least about 2.5:1.
 6. The cassette of claim 1, whereinsaid first and second side walls include a length and compriseperforated walls constructed with spaced apart ribs, said ribs of saidfirst side wall being offset along said length with respect to said ribsof said second side wall.
 7. The cassette of claim 1, furthercomprising: a flange extending along upper portions of at least two ofsaid side walls, said flange including depressions configured toregister with detents in a frame during a tissue embedding process. 8.The cassette of claim 1, wherein said bottom wall includes a centralarea and comprises a perforated wall constructed with spaced apart ribs,said ribs angling toward said central area.